In the study of cell populations associated with disease states it is frequently found that only a subpopulation of available susceptible cells actually exhibit the morbid phenotype. In infectious diseases, the proportions of cells which are passively or actively infected may be very low, and the disease caused by the infectious agent may go unnoticed clinically even though an infected individual can transmit the agent to others. Examples of such infectious agents are clammy, protozoans, certain bacteria, and many viruses.
Amongst viruses, the human immunodeficiency virus (HIV) is known to have an extremely long latent period before the onset of the clinical symptoms known as AIDS. Latency may extend to several years during which the infected individual is capable of transmitting the virus to others through intimate contact, sharing of intravenous injection apparatus, or through donation of blood products.
HIV infection is specific for thymus-derived lymphocytes (T cells), and in particular the subset T cells having immune helper function. These T cells possess highly specific HIV receptors on their surfaces to which the virus attaches to gain entry to the cell. Monoclonal antibodies, grouped generally in the CD4 cluster, see Leukocyte Typing III, Ed. A. J. McMichael, Oxford University Press, 1987, and specific for the HIV receptor, have been isolated heretofore (see Kung et al., U.S. Pat. No. 4,381,295). A signal molecule can be attached to such antibodies which binds selectively to those cells expressing the receptor antigen thereby identifying the helper T cell subpopulation. Quantitation of cell numbers of such lymphocyte subpopulations may conveniently be carried out in a flow cytometer.
In normal individuals approximately 50 percent of peripheral T cells are helper cells. In HIV infected patients, this proportion declines sharply because the virus is cytotoxic to helper T cells. In latent infections or early in the course of clinical disease, the proportion of the helper T cell population actually containing virus, in either lytic or latent phase, may be very low, even to the order of 1 to 1000. This means that in such patients, a 2 ml sample of blood may contain only one or a few copies of the virus or its genome. At his stage of infection, no antibodies to viral proteins can be detected, even with the most sensitive immunological techniques available. There is a great danger that individuals at such early stages of infection may transmit the virus in donated blood without the virus being detected by conventional screening methods.
The most sensitive immunological techniques are capable of detecting antibody by HIV at minimally 21 days post-infection. A variety of immunoassays for detection of HIV have been described including enzyme-linked immunoassays (ELISA), immunodiffusion assays, radioimmunoassays (RIA), and the classical Western blot. Also a number of distinct assay strategies have been developed. One group of assays utilizes HIV viral antigens, particularly viral protein containing epitopes in conserved domains, bound covalently to a solid matrix. The matrix-bound enzyme is contacted with a serum sample, and any anti-antigen antibodies contained therein bind to antigen. In the typical sandwich assay, antiserum raised in a heterologous species against human antibody antigens conjugated to an enzyme (ELISA), fluorescent molecule, or other signal generating substance, is then reacted with the washed matrix-bound antigen-anti-antigen complex. The signal emitted by the signal generating substance is typically a chromophor, fluorescent signal, beta or gamma radiation, or other such measurable emission.
Alternatively, analysis of serum antibodies may be obtained by Western blot consisting of gel electrophoresis of viral proteins, electrotransfer of the proteins to blotting paper, followed by reaction with antisera, and color development of the individual protein bands. The Western blot analysis is employed on a confirming test by blood banks in blood screening procedures. For a general review of the various immunological methods available, see Stites et al., Basic & Clinical Immunology, Appleton & Lange, 1987.
There have been many attempts in the prior art to make detection of serum antibodies to HIV or other low concentration targets more sensitive and selective. The two major approaches have been target amplification and signal amplification. In signal amplification, the object is to couple a very low level signal event to a large number of subsequent secondary signals which can be detected and quantified. It is apparent that this coupling must be highly specific so that background secondary signals do not proportionally increase. One such signal amplification system takes advantage of the extremely high affinity of avidin for biotin. A large number of biotin molecules can be covalently coupled to an antibody specific for viral antigens. When reacted with fluorochome-coupled avidin a large complex is formed having unusually brilliant fluorescence. Another system utilizes a mixture of monoclonal antibodies conjugated to a signal generating substance, each individual antibody type being specific for a different structurally distinct epitope. The theory is that a greater number of signal generating antibody molecules will bind to antigen if there are no overlapping specificities.
Another approach is to target nucleic acid sequences of the virus with a homologous nucleic acid probe coupled to a signal amplification system. Under renaturing conditions the viral RNA (or denatured DNA) anneals to the complementary sequence of an oligonucleotide probe. Detection of the hybrid is afforded by signal generating substances covalently conjugated to the probe. Applicable to his approach are the enzyme proteolyzes a zymogen which then acts upon a substrate to generate a measurable signal. Many of the variations in such techniques are described in Lelie et al., Detection of HIV Infection Using Second-Generation HIV Assay, IV International AIDS Symposium, Stockholm, 1988.
The second major approach involves target amplification in which the target interacting with the signal-generating entity is itself multiplied in number. Since proteins cannot replicate, target amplification inherently requires a nucleic acid sequence, and an enzymatic system which can replicate the target sequence in vitro. One such target amplification technique is disclosed in U.S. Pat. No. 4,683,195 (Mullis et al.) and U.S. Pat. No. 4,683,202 (Mullis), and is called polymerase chain reaction (PCR) amplification.
In PCR, a mixture of nucleic acids containing a DNA sequence in a small quantity is heated to denature double stranded DNA. Primers consisting of a oligonucleotide capable of mediating DNA synthesis from a single stranded template is added under conditions which favor annealing of the primers to their specific complementary sequences. A thermostable DNA polymerase is added, and an extension reaction proceeds at 72.degree. C. in the presence of deoxynucleotide triphosphates, adenosine triphosphate and cofactors. The reaction is run at high temperatures to avoid non-specific binding of primer to non-homologous sequences. Under these stringent conditions fidelity of polymerization to the desired sequences is very high.
After polymerization is complete, the mixture is again heated to denature the double stranded DNAs, and the extension reaction is repeated. Such repetition of extension polymerization may occur several times until the target sequence is amplified in numbers sufficient to detect by any of the signal-conjugated probe assays described hereinabove.
In a second target amplification scheme called TAS, a first primer oligonucleotide or oligonucleotide containing an RNA transcriptase promoter-binding sequence is annealed to the target sequence and extended by DNA polymerase or reverse transcriptase. Following melting, a second primer complementary to the newly formed oligomer in a region distal to the first primer binding sequence is added, annealed, and extended. The resultant duplex DNA oligomer thus has a sequence flanking the target region and containing a transcriptional promoter. Addition of RNA transcriptase, in the presence of oligonucleotide triphosphates, adenosine triphosphate, and cofactors institutes transcription in vitro yielding up to 1000 copies of the target sequence. The TAS methods have been disclosed in WO 88/01050 (Berg et al.).
In a variation of TAS, RNAse H is added to the reaction mix. RNAse H specifically catalyzes the step-wise hydrolysis of RNA bases in an RNA-DNA duplex, so that after a cDNA strand has been synthesized with reverse transcriptase the RNAse digests the RNA strand of the duplex to permit synthesis of the second complementary DNA strand by DNA polymerase. It will be apparent that since a heating step to melt the DNA-RNA duplex is unnecessary for cyclization of the reaction, the entire amplification can be performed in a single incubation. The disadvantages of the TAS and 3SR methods, compared to PCR is the lesser degree of stringency because of non-specific primer bonding at the lower temperature.
Finally, target amplification can be carried out in a ligase-mediated procedure. In this procedure, complementary primer sets form adjacent hybrids on both complementary strands of the target. Ligase then joins the primers together at the nick separation, after hybridization. The ligated double primer can then act as a template for further ligation of primers in a subsequent melting and rehybridization step.
After the target amplification, the nucleic acids are ordinarily extracted and the amplified sequences are detected by the procedures set forth hereinabove. It should be emphasized that the known procedures of the prior art are heterogeneous, that is, they require multiple steps in which the DNA is first hybridized to a signal generating probe, followed by a step in which the unhybridized probe is separated from hybridized probe. ordinarily different sets of reagents are required for signal generation than for probe hybridization and separation. A completely homogeneous method for detection of amplified nucleic acids without a separation step is unknown in the prior art.